The goal of the proposed research is to exploit the simple cleavage and ligation reactions of hairpin ribozymes to assess the dynamics and equilibria of RNA conformational transitions in a biological context. RNAs are critical components of the biological machinery responsible for maintenance and expression of genetic information, providing potential therapeutic targets and reagents for therapeutic intervention. RNA assembly reactions and exchange among alternative RNA structures play key roles in transcription, translation, viral replication and RNA processing reactions. Quantitative analyses of specific RNA folding events in most RNA-mediated biological processes are complicated by the participation of many components in complex pathways. On the other hand, RNA enzymes report on assembly of functional RNA structures directly through their catalytic activity. Studies of RNA folding using RNA enzymes as model systems in vitro have produced a wealth of information regarding the kinetics and equilibria of individual steps of RNA- mediated reactions and the influence of small molecules, RNA binding proteins, and RNA transcription on RNA assembly pathways in simple test tube reactions. Our goal is to learn how the principles that have been elucidated through in vitro studies translate to the behavior of RNAs in the complex environment of a cell. The hairpin ribozyme is an especially valuable model for RNA structure-function studies in vivo because ribozyme variants have been designed so that intracellular cleavage activity monitors specific RNA conformational transitions along the reaction pathway. Specific goals are 1) to use inter-molecular ribozyme cleavage activity to probe the specificity, kinetics and equilibria of RNA complex formation in vivo, 2) use ribozyme variants with the potential to adopt defined nonfunctional secondary structures to distinguish rapid conformational exchange and delayed folding models of intracellular RNA assembly, 3) determine how post- translational RNA re-folding pathways differ from co-transcriptional folding and 4) characterize the kinetics and equilibria of small ligand-induced RNA conformational transitions in vivo. Mechanistic insights gleaned using this simple system to report on RNA structure and dynamics in specific biological contexts will help illuminate mechanisms of more complex RNA-mediated reactions and provide a rational framework for the development of RNA-based therapeutics.